Summary of work: UV irradiation of S phase-synchronized cells causes delays in completion of S phase followed by an extended G2 arrest and, in some cases, apoptosis. We used genetically-related Chinese hamster ovary cell lines proficient or deficient in DNA repair to determine whether persistence of UV-induced photoproducts following S phase irradiation was directly involved in inhibition of DNA replication and in induction of G2 arrest and apoptosis. We found that nucleotide excision repair activity is neither enhanced nor inhibited during S phase. Further, the effects of UV irradiation on S and G2 phase progression are magnified in repair-deficient cells, indicating that these effects are initiated by persistent DNA damage and not by direct UV activation of signal transduction pathways. Moreover, persistence of (6-4) photoproducts inhibits DNA synthesis much more than persistence of cyclobutance pyrimidine dimers, which appear to be efficiently bypassed by the DNA replication apparatus. Apoptosis begins approximately 24 hr after UV irradiation of S phase-synchronized cells, occurs to a greater extent in repair-deficient cells, and correlates well with the inability to escape from an extended late S/G2 phase arrest. The human CSB gene, mutated in Cockayne's syndrome group B (partially defective in both repair and transcription) was previously cloned by virtue of its ability to correct the moderate UV sensitivity of the CHO mutant UV61. To determine whether the defect in UV61 is the hamster equivalent of Cockayne's syndrome, the RNA polymerase II transcription and DNA repair characteristics of a repair-proficient CHO cell line (AA8), UV61, and a CSB transfectant of UV61 were compared. In each cell line, formation and removal of UV-induced CPDs was measured in the individual strands of the actively transcribed DHFR gene and in a transcriptionally inactive region downstream of DHFR . AA8 cells efficiently remove CPDs from the transcribed strand, but not from either the nontranscribed strand or the inactive region. There was no detectable repair of CPDs in any region of the genome in UV61. Transfection of the human CSB gene into UV61 restores the normal repair pattern (CPD removal in only the transcribed strand), demonstrating that the DNA repair defect in UV61 is homologous to that in Cockayne's syndrome (complementation group B) cells. However, we observe no significant deficiency in RNA polymerase II-mediated transcription in UV61, suggesting that the CSB protein has independent roles in DNA repair and RNA transcription pathways. In response to UV irradiation of mammalian cells, the protein proliferating cell nuclear antigen (PCNA) forms an insoluble complex with nuclear substructures. This complex can be detected by immunofluorescence and western blot analysis within 30 min after UV irradiation. We have studied the role of nucleotide excision repair (NER) and its subcomponent, transcription coupled repair (TCR), in PCNA complex formation. PCNA complex formation was studied in genetically related hamster cell lines that differ only in their capacity to perform NER. The hamster cell lines UV5 and UV24, which are homologs of the human DNA repair mutants xeroderma pigmentosum (XP) groups D and B, are completely deficient in NER. The hamster cell line UV61 is deficient in TCR of UV induced pyrimidine dimers, and is homologous to the human DNA repair mutant Cockayne syndrome (CS) complementation group B (CS-B). In the NER deficient cells, UV5 and UV24 cells, the PCNA complex was not detectable within 30 min after UV. When the UV5 cells were transfected with the human XPD gene, the PCNA complex formation was restored to normal. In the TCR defective UV61 cells, the rate of PCNA complex formation was intermediate between normal and NER deficient cells. This defect in UV61 cells was complemented by transfection of the human CSB gene. We conclude that efficient PCNA complex formation induced by UV irradiation is dependent on both the genome overall repair of 6-4 photoproducts and the TCR of pyrimidine dimers in hamster cells. The complex formation occurs only in non-replicating cells and is not affected by pretreatment with aphidicolin.